Stabilized gas-in-liquid emulsions are useful in a variety of fields. For ultrasonic imaging, the most common contrast agents contain many small bubbles. Gas-filled microbubbles are a proven contrast agent in ultrasonic imaging. Their difference in density makes them an excellent means for scattering ultrasonic waves. Moreover, air injected microbubbles travel with intracardiac velocities similar to red blood cells making them particularly useful in echocardiography. In therapeutic applications, drug and targeting agents may be combined with bubbles, infused in a patient, and these preferentially gather at the disease site. Ultrasound energy could then be used to disrupt the bubbles and to release the drug locally. The ultrasound could also be used to disrupt the bubbles, to induce acoustic activation, sonoporation, inertial cavitation, and the like, in order to permeabilize tissue so that the drug is released locally and the cellular uptake and efficacy of the drug enhanced.
Bubbles may also be used to accelerate the heating cycle of high intensity frequency ultrasound (HIFU) tumor ablation treatments, reduce treatment duration, and thus reduce patient trauma and expand potential applications. The bubbles may even be used to reduce the energy required for ultrasound systems designed to lyse fat cells through cavitation. The term “cavitation” defines a physical process whereby tiny bubbles present in the liquid are made to grow and collapse with great force. This occurrence produces violent pressure changes in the sonicated liquid at multiple microscopically spaced volume elements within the liquid. These pressure changes, which may be thousands of atmospheres in magnitude, break up any clusters of cells and may disintegrate the cells themselves, if the cavitation is sufficiently intense. Recently, microbubbles have been used with low frequency ultrasound to intentionally cause cavitation in tissue.
It is desirable that microbubbles used in the above applications have a mean average diameter of about 1 to 10 microns. Generally, this is because bubbles in excess of 10 microns in diameter are short lived as they are quickly absorbed by the vascular bed of the lungs. Bubbles less than 1 micron may not achieve the desired increased backscatter or increased rate of attenuation of sound energy, or sufficiently alter the speed of transmission of ultrasonic waves as to be useful for therapeutic means. Bubbles less than 1 micron may also not induce the desired pressure changes when sonicated as to cause significant cavitation in order to permeabilize tissue for drug treatment or disrupt cellular tissue. It is also desirable for a bubble solution to be stable enough for its intended use inside a human or animal subject. When used in imaging the microbubbles should not dissipate immediately after injection and last at least one circulatory pass inside a human or animal subject. The bubble solution should also retain enough stability after injection into tissue as to be suitable target of ultrasonic waves to cause the necessary cavitation of internal tumors or tissue, or disruption of cells.
Various methods for generating microbubbles have been devised and several patents have been published for devices and methods of generating sufficiently stable microbubbles of an optimal size and consistency. U.S. Pat. No. 5,352,436 to Wheatley et al., incorporated herein by reference, discloses a mixture of, and the process of preparing, stabilized gas microbubbles formed by sonication. The mixture is created by mixing a solvent, a first surfactant, and a second, dispersible surfactant. Preferably the first surfactant is substantially soluble and non-ionic, such as polyoxyethylene fatty acid esters including commercially available TWEEN. Preferably, the second dispersible surfactant may be partially or fully soluble in the solvent, is non-ionic, and is a sorbitan fatty acid ester including SPAN which is a commercially available dry powder. Microbubbles are generated in the mixture by exposing the mixture to ultrasound sonication for about 1 to about 3 minutes at power levels between about 140 to 200 watts. The mixture is permitted to separate into a dense solvent layer or aqueous lower phase, an intermediate layer or less dense phase comprising substantially all the microbubbles having a mean diameter less than about 10 microns and an upper layer comprising substantially all of the microbubbles having a mean diameter greater than about 10 microns. The intermediate layer is then separated from the upper and lower layers using a separatory funnel and washed with a saline solution.
While the microbubbles in Wheatley et al. were reported to remain stable for three days, it has been observed that each required separation cycle—at least once to form the first intermediate layer and second when washed—requires a substantial time period (e.g. 10 to 15 minutes for each period) for gravity to collect the layer of surfactant-stabilized microbubbles above the solvent or lower layer. Unless temperature controlled storage is available to store the microbubble solution for successive treatment it would be preferable to create microbubbles during the treatment cycle. Moreover, sonication requires noise levels which are unacceptable for use during patient treatment. As disclosed by U.S. Pat. No. 4,957,656 to Cerny et al., incorporated herein by reference, the vibration frequencies of sonication equipment can vary over a considerable range, such as from 5 to 40 kilohertz (kHz), but most commercially available sonicators operate at 20 kHz or 10 kHz, performing well at these ranges for generating microbubbles. The primary drawback in using sonication for generating microbubbles has been the large size and weight of the processing equipment. Commercial sonicators are large, heavy, tabletop devices that require power from a standard outlet and way up to or over a kilogram. It is also well known that the noise generated from the sonicator apparatus in these ranges is objectionable during patient treatment, especially at or below 20 kHz. Thus, when microbubbles are to be formed through sonication the microbubble solution is prepared well in advance of its use in treatment.
Various systems and methods have been proposed for creating microbubbles during the treatment of a patient. U.S. Pat. No. 6,575,930 Trombley, III et al., incorporated herein by reference, is directed to a system for dispensing a medium including at least a first container to hold the medium, a pressurizing device, such as a pump, in fluid connection with the container for pressurizing the medium, and an agitation mechanism or device to maintain the components of the medium in a mixed state. The container and pump can be a syringe whereby the method of injecting the multi-component medium includes agitating the medium before or during the injection. Although Trombley, III et al. works well for maintaining the constant bubble source, the device and method does not allow for selectivity in microbubble size. If the right mixture is attained a preferred size may be obtained (e.g. 1 to 10 microns), however, larger bubbles may also be created, and it is impossible to select a specific range of bubbles within the range created by the agitation method.
Attempts have been made to generate microbubbles in a syringe for immediate injection into a treatment area. As explained by U.S. Pat. No. 5,425,580 to Beller, DE-A 3 838 530, and EP-A 0 148 116, all incorporated herein by reference, producing microbubbles in a syringe just before administration to a patient has been achieved by drawing a contrast medium together with air or a physiologically tolerated gas into a syringe, then connecting the syringe by a connector to a second, empty syringe. Vigorous pumping of the medium backwards and forwards between the two syringes produces microbubbles. Beller improves upon this method of generating microbubbles in a syringe by using a mixing chamber disposed between the syringes and having mixing elements in the form of spikes preferably at right angles to the inner wall of the mixing chamber, and a predetermined amount of sterile gas in the mixing chamber, thereby reducing effort required to force the liquid between the syringes to create the microbubble solution.
U.S. Publication No. 2008/0269688 to Keenan, incorporated herein by reference, discloses rotating the syringes about an axis parallel to the earth so that during each half reciprocal cycle there will be a lower syringe and an upper syringe, with the lower syringe being inverted such that is output is upward. When the lower syringe is inverted, the unusable gas containing bubbles greater than 10 microns will migrate upward toward the surface of the solution inside the syringe. These larger bubbles can then be expelled by the lower syringe into the upper syringe leaving the more useful bubble solution in the lower syringe. The process is repeated for the upper syringe by inverting the two syringes for the next half reciprocal cycle. This method has been found to work for most conventional means, however, the process of inverting the solution, waiting for separation, expelling the unusable solution, and then repeating the cycle can take up to 10 minutes before an optimal concentration of bubble solution is available for use in a patient.
It has also been hypothesized that the syringes could be rotated at high speeds to more quickly separate the bubble fluid by centrifugal force. It has been found, however, that a major drawback to using centrifugal force in conjunction with two connected syringes containing a bubble solution will cause any usable bubbles to separate away from the outlet of the syringe. It is widely known that spinning a vessel containing materials of different specific gravities about a central axis will create an outward force associated with the rotation that will move the heavier liquid outward, due to the centrifugal force, while the gas migrates inward toward the central axis. This means that, as the bubble solution separates inside the syringe, an upper layer comprising most of the gas and unusable microbubbles greater than 10 microns will migrate toward to outlet, near the axis, while a dense solvent layer of aqueous solution will migrate the toward the syringe pump, disposed at the outer perimeter of the rotation. An intermediate layer or less dense phase comprising substantially all the microbubbles having a mean diameter less than about 10 microns will migrate toward the middle of the syringe. The syringe must then be removed and properly positioned upright so that gravity will move the unusable gas near the outlet so that it can be expelled by pushing in the plunger of the syringe while the output of the syringe is facing in an upward direction. This process takes time and when the syringe is initially inverted there is an increased risk of mixing the usable bubbles with unusable bubbles. Moreover, there is effectively no way to further separate the lower dense layer of aqueous solution to retain a highly concentrated solution of usable microbubbles having a mean diameter less than about 10 microns.
Thus it can be seen from the relevant art developed that a method and device for generating microbubbles that is flexible enough to supply a wide range of chemistries, quiet and small enough to be used during treatment of a patient, fast, inexpensive, and reliable for storage and shipping and changing of environmental conditions. Moreover, the device and method should efficiently separate a bubble solution and extract a concentrated microbubble solution having microbubbles between 1 and 10 microns in diameter for immediate use in treating a patient.